Alkaline denaturation of β-lactoglobulins. Activation parameters and effect on dye binding site

Waissbluth, Mario; Grieger, R. A.

doi:10.1021/bi00703a035

Cited by 21 publications

(10 citation statements)

References 15 publications

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“…In fact, the changes in ACP with temperature in the range 15-35 °C were found to be small, about 15%, and the experimental error in the actual determination of ACP was as high as 18%. The average value of ACP in the temperature range 15-35 °C comes out to be +2700 cal mol-1 deg-1 which is expectedly higher than the value (1400 cal mol-1 deg-1 below 40 °C) for the process, native -* activated state, calculated from the kinetic data on the denaturation of ovalbumin in absence of any denaturant (see Waissbluth and Grieger, 1974). It is noteworthy that recently Privalov and Khechinashvili (1974) on the basis of their studies on the thermal denaturation of several proteins have concluded that the temperature dependence of ACP is much smaller than found previously.…”

Section: Resultsmentioning

confidence: 80%

Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

Ahmad¹,

Salahuddin²

1976

Biochemistry

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The guanidine hydrochloride induced unfolding of the major fraction of ovalbumin (i.e. A1 which contains two phosphate groups and constitutes about 77% of the total protein) was investigated systematically by difference spectran and viscosity measurements. As judged by the intrinsic viscosity (3.9 ml/g), the native protein conformation is compact and globular. Difference spectral results showed extensive disruption of the native structure by guanidine hydrochloride with and without 0.1 M beta-mercaptoethanol were 31.1 and 27.0 ml/g. These and optical rotation results indicated that the denatured protein existed in a cross-linked random coil conformation in 6 M guanidine hydrochloride alone. Strikingly, in contrast to whole ovalbumin, the denaturation of its A1 fraction by guanidine hydrochloride was fully reversible and obeyed first-order kinetic law under different experimental condit ions of pH, temperature, and the denaturant concentration. The monotonic variation of deltaH for the unfolding of ovalbumin A1 by guanidine hydrochloride with temperature, the coincidence of the two transition curves obtained by measuring two independent properties (namely reduced viscosity and difference in light absorption at 288 nm (or 293 nm) as a function of the denaturant concentration, and finally the adherence of the unfolding as well as refolding reactions to first-order kinetic law suggested that the transition of ovalbumin. A1 can reasonably be approximated by a two-state mode. Analysis of the equilibrium data obtained at pH 7.0 and 25 degrees C according to Aune and Tanford (Aune, K.C.,and Tanford, C. (1969), Biochemistry 8, 4586) showed that 12 additional binding sites for the denaturant with an association constant of 1.12 were freshly exposed by the unfolding process and that the native protein was marginally more stable (approximately 6 kcal/mol) than its unfolded form even under native condition. The temperature dependence of the equilibrium constant for the unfolding of ovalbumin A1 by guanidine hydrochloride which was studied in the range 10-60 degrees C at pH 7.0 can be described by assigning the following values of the thermodynamic parameters for the unfolding process: deltaH = 52 kcal/mol at 25 degrees C; deltaS = 153 cal deg-1 mol-1 at 25 degrees C; and delta Cp = 2700 +/- 400 cal deg-1 mol-1.

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Section: Resultsmentioning

confidence: 80%

Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

Ahmad¹,

Salahuddin²

1976

Biochemistry

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“…This transition, which occurs between pH 9 and pH 13, has been previously identi®ed as the baseinduced denaturation of b-lactoglobulin. 16,18,20 It should be noted that the transition also results in ®nal disruption of any remaining native-like dimeric structures into unfolded monomers. The near UV CD spectra of the protein (Figure 3) suggest that the base-induced denatured state of b-lactoglobulin at pH 12.51 (^) lacks rigid tertiary structure present in its native state (below pH 9).…”

Section: The Base-induced Denaturationmentioning

confidence: 99%

“…where b So is the coef®cient of adiabatic compressibility of the solvent. The volumetric and ultrasonic velocimetric experiments have been performed at least three times with the average values of [u] and v being used in equation (20).…”

Section: Determination Of the Partial Specific Adiabatic Compressibilitymentioning

confidence: 99%

Characterization of pH-induced transitions of β-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies 1 1Edited by C. R. Matthews

Taulier

Chalikian

2001

Journal of Molecular Biology

208

178

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“…It should be noted that all transitions that take place between pH 2 and pH 9 do not cause any appreciable changes in the native-like bbarrel conformation of BLG (Blanch, Hecht, & Barron, 1999). By contrast, above pH 9, BLG undergoes an irreversible, base-induced unfolding transition with global disruption of both secondary and tertiary structures (Groves, Hipp, & McMeekin, 1951;Tanford, Bunville, & Nozaki, 1959;Waissbluth & Grieger, 1974). Studies have found that BLG at 100 mM is dimeric at 25 C over a pH range of 2.5e7.5.…”

Section: Introductionmentioning

confidence: 99%

Characterising the secondary structure changes occurring in high density systems of BLG dissolved in aqueous pH 3 buffer

2015

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a b s t r a c tThis study looks at the influence of reduced levels of hydration as a driving force for transitions in the secondary structure of hydrated proteins. A simple protein-water system was used to study the conditions of typical protein-rich dairy food systems at a fixed pH level, salt content, and temperature. Freezedried beta-lactoglobulin (Type A) from bovine milk was dissolved directly into two different buffer systems over a wide range of concentrations between 1 mg/ml (~54 mM) and 200 mg/ml (~0.01 M) but at a fixed pH level, pH 3. Circular dichroism (CD), attenuated total reflectance Fourier transform infrared spectroscopy (ATR FTIR), and thioflavin T (ThT) Assay fluorescence spectroscopy were used to measure changes in the secondary structure with respect to protein solution concentration at 20 C. The findings of all of the techniques indicate that the majority of the secondary structure changes occur within the low protein concentration regime (i.e. <50 mg/ml) before a critical aggregation threshold. Dimerisation, formed by besheet cross-linking, is the likeliest mechanism of aggregation. The formation of dimers however counters the current assumption that at pH 3 only monomers exist; rather it seems there is a gradual evolution of the monomeric unfolded state with increasing concentration occurs yielding a bsheet rich refolded aggregate. Most interesting is the low concentration region (i.e. between 1 mg/ml and 40 mg/ml) where most secondary structural alterations are found to occur; before physical crowding effects are possible. The results indicate that BLG has a limited solubility even in a low concentration regime.

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Alkaline denaturation of β-lactoglobulins. Activation parameters and effect on dye binding site

Cited by 21 publications

References 15 publications

Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

Characterization of pH-induced transitions of β-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies 1 1Edited by C. R. Matthews

Characterising the secondary structure changes occurring in high density systems of BLG dissolved in aqueous pH 3 buffer

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